Biosensing devices for detection of biological or chemical components have become widely used in many applications such as medical diagnostic laboratories, point-of-care settings, and field work. The utility of such devices in point-of-care testing has grown rapidly due to the benefits of providing portable and immediate results to assist in clinical management decisions of early detection and disease screening. Biosensor applications have advanced with recent developments in biomolecular chemistry technologies which provide reagents with improved selectivity and affinity to specific biological targets, such as specific disease markers expressed by proteins, antibodies and nucleic acid fragments or DNA and the like.
A primary function of a biosensor is to provide an output corresponding to the quantitative detection of the presence and/or relative abundance of a specific target biomolecule in a given analyte. The analyte can be presented to the biosensor either in-vivo or in-vitro to the source or patient. Enzyme-linked immunosorbent assay tests (ELISA) are one category of biosensors, in which a target biomolecule can be detected for presence and quantified within an analyte volume. In general, a glass plate is prepared with an array of identical analyte volumes or wells. Within each of the wells a specific bioreceptor is attached to the inner surface of the glass wells with a known concentration. The bioreceptors are typically either an antibody (Ab) or an antigen (Ag)—and the Ag-Ab pair is preselected with criteria for high selectivity and affinity to a target biomolecule under test. An analyte comprising the material under test is then titrated in each well to a predetermined concentration matrix and allowed to react with the specific bioreceptors. The analyte comprises the complementary substance to the particular bioreceptor that is used. If the target species is present in the analyte it will be immobilized and bound to the bioreceptor. Bioreceptors can be developed to produce a physical change indicating the binding event or a subsequent reagent can be passed over the wells to reveal the result.
In general, the ELISA sequence is engineered so that an optical indicator results when a successful binding event occurs. The degree to which the target within the analyte binds to the bioreceptors is detected optically as either a change of color or optical density. The magnitude of the optical response is directly proportional to and representative of the number density of the target biomolecule within each well. The optical response of each of the wells is typically measured using an optical sensor that measures the optical intensity or power due to the absorption or fluorescence signal. The optical sensor may also be tuned to a specific wavelength or wavelength range. The optical sensor may also capture an image of the entire plate comprising an M×N array of wells (typically, M and N are integers characterizing the assay configuration of the wells with standard sizes of M×N=96, 384 and 1536 wells). For example, the test plate optical response can be directly mapped using a digital charge-coupled device (CCD) array. Furthermore, if more than one bioreceptor is utilized within each well it is possible to produce a specific and unique wavelength response for each particular antigen-bioreceptor event type, thereby producing improved throughput for the diagnostic test via the use of optical multiplexing. A wavelength selective sensor is beneficial for this purpose. The two main types of optical responses used to probe the antigen-photoreceptor binding event are: (i) optical absorption and (ii) optical excitation fluorescence. Others are also possible, such as, electrically stimulated fluorescence. It is well known in prior art that optical techniques enable large ensembles of such arrays to be processed with high throughput. The trade-off to throughput and array size is typically due to the lower limit of sensitivity for detection of the target binding event.
The use of optical interrogation of the described test array necessarily requires an optical sensor to be spaced at a distance from the plate to enable collection of light or optical imaging of the well or array. That is, imaging and focusing optics are required to probe the regions of interest and provide directed optical energy to a receiving optical sensor for measurement. The sensitivity of the optical detection process is therefore limited by the analyte volume contained in each well, the optical cross-section presented by the binding event through absorption or fluorescence, the transparency of the materials comprising the array, and the etendue limit of the optical system used.
Yet a further class of biosensor measurement systems require the quantification of the unique optical wavelength absorption spectrum or fluorescence emission spectrum of a particular analyte. So-called, label-free detection of target biomolecules is becoming of increasing utility for biomolecular sensors. Resolving the wavelength response of target biomolecule species within an analyte further requires the use of at least one of a wavelength spatially dispersive element, such as, a refractive prism or diffractive grating. These dispersive wavelength spectroscopic methods further increase the complexity of the biosensing apparatus and reduce the light collection to the optical sensor, thereby increasing the optical lower limit of sensitivity.
Biosensing of biomolecules for medical research, such as proteins and DNA, may also be characterized by their ultraviolet spectral absorption or fluorescence spectrum. In such devices, a sample is subjected to ultraviolet light and the output sensed with a detector. In some devices, a specific wavelength is selected from the source, and the sample is swept with varying wavelengths to characterize the response of the sample. In other devices, the sample is subjected to a broad spectrum and a dispersive or wavelength selective filter is used to select a particular wavelength to be analyzed by the detector. In either type, a single detector is used, which is broadly sensitive. In addition, the light collection optical path requires distance between the analyte and the detector, which limits the sensitivity of the device.
As biosensors continue to be more widely used, there is a continuing need for improved functionality and lower cost devices.